Identification of bacillus thuringiensis based on biologycal tests and novel biomarkers

INTRODUCTION

Preface

The rising demand for food due to a growing population has led to a significant increase in the use of chemical fertilizers in agriculture However, this excessive reliance on chemical fertilizers negatively affects the agricultural ecosystem Consequently, research is shifting towards alternatives that enhance crop productivity while preserving soil health Biopesticides, derived from natural substances, offer a non-toxic and eco-friendly solution for pest control Among these, Bacillus spp., particularly B thuringiensis, has gained popularity in the biopesticide market for its effectiveness against pests in economically vital crops.

Bacillus thuringiensis (Bt), part of the Bacillus cereus group alongside Bacillus cereus, B mycoides, and B anthracis, is a gram-positive, spore-forming bacterium known for its Cry gene This gene encodes a toxic crystalline protein (Cry protein) that targets various pests and diseases affecting plants Recognized as an environmentally friendly biopesticide, Bt plays a significant role in pest and nematode management strategies.

Bacillus thuringiensis (Bt) produces various virulence factors that penetrate the intestinal epithelial cells of insects Key among these are the δ-endotoxins, which are proteinaceous crystals formed during sporulation These delta-endotoxins exhibit targeted insecticidal properties against several insect orders, including Coleoptera, Diptera, Lepidoptera, Hymenoptera, and Hemiptera, as well as certain invertebrates.

Identifying microorganisms within closely related groups poses significant challenges Traditional methods such as biochemical tests, fatty acid profiling, and DNA–DNA hybridization are often time-consuming, making them unsuitable for rapid identification For instance, distinguishing between Bacillus thuringiensis (Bt) and Bacillus cereus (Bc) based solely on morphological characteristics is not feasible.

2 utilization of organic compounds (Baumann et al., 1984; Logan & Berkeley, 1984; Priest et al., 1988), characterization of cell content of fatty acids (Vọisọnen et al.,

1991) or sugars (Wunschel et al., 1994), enterotoxin production (Damgaard et al., 1996a; Hansen & Hendriksen,1997a) Likewise, genotypic differentiation of Bt and

DNA homology analysis, 16S rDNA sequencing, and the analysis of the 16S-23S internal transcribed sequence have proven inadequate for distinguishing B cereus from B thuringiensis due to their high genetic similarity (Kaneko et al., 1978; Ash et al., 1991; Peng et al., 2015) This highlights the urgent need for accurate identification methods for B thuringiensis Recent research by Chelliaha et al (2019) identified eight genes that can effectively differentiate B thuringiensis from the B cereus group, with GroEL, gyrB, and XRE showing particularly promising results Supporting this, Wei et al (2019) confirmed that targeting the XRE gene is an efficient molecular approach for distinguishing B thuringiensis from B cereus in various samples.

In Vietnam, research on Bacillus thuringiensis primarily emphasizes the analysis of morphological characteristics, the detection of toxic genes, and 16S rDNA sequencing However, the simultaneous application of various conventional and molecular techniques for the detection of Bt remains limited This study aims to address these gaps in research.

Bacillus thuringiensis based on biological test and novel biomarkers was proposed.”

Objectives and Requirements

Identification of Bacillus thuringiensis using biological test and species- specific PCR

1.2.2 Requirements o Identification of isolated Bt based on morphological and biochemical characteristics o Identify the appropriate method for total DNA extraction o Identification of isolated Bt based on species-specific PCR analysis o Confirmation of the presence of Bt using crystal protein staining.

LITERATURE OVERVIEW

Overview of Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is a natural bacterium found in various environments, particularly in three biological niches: insects, plants, and soil (Barjac D H, 1981) It can be easily cultivated in laboratory settings with minimal fertilizers While Bt spores have a long lifespan, they do not germinate and grow like vegetative cells in natural soil.

Bt is a gram-positive, rod-shaped, 3-6 μm -sized bacteria that may be isolated or chained Aerobic or facultatively anaerobic bacteria that are motile due to surface-growing cilia

As a member of the genus Bacillus, Bt has the potential to create endospores to battle unfavorable situations like nutrient deficiency, high temperature, and drought

The Bt life cycle consists of three stages:

 Nutritional body: rod-shaped with two obtuse ends, often standing alone or capable of forming chains Reproduces via horizontally splitting

 Spore cyst: Mature, elongated, ovoid cell bigger than the vegetative body; one end creates round (oval) spores, and the other develops crystals

 Spores and crystals: As the sporangium matures and explodes, spores and crystals are released

Spores are dormant bacterial life forms resistant to heat, radiation, and chemicals

Figure 2.1 Spore–crystal morphology of Bacillus Thuringiensis

Notes: (B) bipyramidal; (C) cubic; (S) spherical; (I) irregularly shaped spherical;

Source: ( https://www.researchgate.net/profile/Ugur-Azizoglu-

The Bt crystal is a protein measuring approximately 0.6 x 0.02 μm, constituting about 25% of the cell's dry mass It exhibits various forms, such as pyramidal, oval, cubic, and indeterminate In vegetative cells, spores and crystals are typically found in close proximity, and they are released together when the cell breaks down (Ngo Dinh Binh, 2005; Barjac, 1981).

Bt does not ferment arabinose, xylose, or mannitol but produces acid in glucose-rich environments It can hydrolyze starch and reduce nitrate to nitrite When cultured in a medium containing 0.001% lysozyme and 7% NaCl at pH 5.7, a reaction occurs with egg yolk, and there is no need for citric acid or sulfate desalination.

Bt has the potential to thrive in temperatures ranging from 15 to 45 o C The ideal temperature is between 20 o C and 30 o C

The ideal pH for Bt growth is 7, although the pH of the environment does not influence its development Bt is capable of converting hydrocarbons into organic acids and carbon dioxide through the Embden-Meyerhoff-Panas cycle (Ngo Dinh Binh, 2005).

Bt is subdivided into various subtypes depending on the features listed below

 The capacity to produce lecithinase enzyme

 Structure of crystals and insect pathogenicity

 The somatic cell agglutination response with matching serums

In 1962, De Barjac and Bonnefoi introduced a serological method for classifying Bt bacteria, which was later refined by Lauren and Thiery in 1996, establishing it as a reliable classification technique Since 1982, the Bt international center at the Pasteur Institute in Paris has been endorsed for use by research facilities worldwide (Ngo Dinh Binh et al., 2002; Ngo Dinh Binh, 2005).

In the early 1960s, the classification of Bt subspecies was initiated through serological examination of flagella (H) antigens (de Barjac & Bonnefoi, 1962), later enhanced by morphological and biochemical criteria (de Barjac, 1981a) Initially, only 13 Bt subspecies were recognized, all of which targeted Lepidopteran larvae However, the discovery of additional subspecies affecting Diptera (Goldberg & Margalit, 1977), Coleoptera (Krieg et al., 1983), and potentially Nematoda (Narva et al., 1991) significantly broadened the diversity of subspecies and their host range.

67 subspecies based on flagellar H-serovars had been discovered by the end of

1998 (Table 2.1) Serovariety lists may be acquired at the Pasteur Institute's reference center in France (Unité des Bactéries Entomopathogènes, Institut Pasteur, Paris, France)

Table 2.1 Current classification of 67 Bacillus thuringiensis subspecies based on their flagellar (H) antigens

Bt toxicity and crystalline protein mode of action

Bt has four types of toxins: three exotoxins and one endotoxin

Exotoxin α, a phospholipase or lecithinase enzyme, targets specific leaf-eating bees and is secreted prior to crystal and spore formation This low molecular weight toxin is thermally unstable, water-soluble, and released during cell disintegration It is particularly toxic to insects with a gut pH ranging from 6.6 to 7.2, causing harm by breaking down phospholipids in their intestinal tissue, leading to significant toxic effects (Toumanoff, 1953).

Exotoxin β is a heat-stable, water-soluble toxin with a low molecular weight of 707 to 850 kDa that inhibits nucleases and RNA polymerases, thereby disrupting RNA production It is effective against a variety of pests, including scaly insects, beetles, and diopterans, particularly targeting young caterpillars with slowed molting metabolism and adults emerging from larvae that have ingested the toxin Research by Brian Federici and Dongwu indicates that the insecticidal efficacy of Bt preparations is only 20% when solely using exotoxins, but this effectiveness can increase to as much as 75% when the toxin is included.

Exotoxin γ is a water-soluble toxin with a low molecular weight that is sensitive to temperature, light, and air As a member of the phospholipase family, it affects phospholipids and releases fatty acids Notably, this toxin becomes inactive when exposed to temperatures of 60 °C or higher for 10 to 15 minutes.

Toxic crystallized protein is responsible for Bt's capacity to target and kill insects

Endotoxin δ, a highly toxic crystal, exhibits the strongest insecticidal properties This protein consists of 1180 amino acids, predominantly glutamic and asparagine, which together make up over 20% of its composition and contribute to its low isoelectric point The crystal's insolubility is influenced by the presence of cysteine, which constitutes less than 20% of the total amino acids, along with other amino acids such as arginine, threonine, and isoleucine.

The crystalline protein contains various components, including carbohydrates, which may merely be byproducts of the spore generation process Additionally, endotoxins are primarily composed of carbon (C) and other chemical elements.

H, O, N, and S Ca, Mg, Si, Fe, and trace amounts of Ni, Ti, Zn, Al, Cu, and Mn are also present However, element P is almost nonexistent

Endotoxin δ is characterized by its insolubility in organic solvents and its solubility solely in alkaline conditions, specifically at pH 11.5 It is also water-insoluble and exhibits heat stability, maintaining its activity even after being cooked at 65 °C for one hour However, it loses its toxicity when subjected to higher temperatures.

100 o C for 30-40 minutes At an excessively high or low pH, the crystal loses its capacity to respond as an antigen and becomes inactive

Figure 2.2a 3D structure of Cry 2Aa protein

The study by Morse et al (2001) reveals that the structure of the Cry2Aa protein indicates an unexpected receptor binding epitope The protein is organized into three distinct regions: region I (purple), region II (blue), and region III (cyan) Additionally, the analysis highlights the presence of hydrophobic regions in cyan, blue, and purple, along with white hydrophobic areas and red N-tails, emphasizing the complexity of the Cry2Aa structure.

2.2.2 Action mechanism of crystalline proteins on insects

Different Bt strains have distinct insecticidal properties The insecticidal capability of Bt species is only shown when insects consume the poisonous crystals

Insect larvae possess a high alkaline pH and a unique protease enzyme system in their gut, which facilitates the dissolution of poisonous crystals When ingested, these crystals are hydrolyzed into δ-endotoxin, specifically the proteotoxic 130kDa and 70kDa proteins This toxin then binds to the epidermal cells of the insect's gut, creating openings that allow ions and water to enter, leading to cell expansion and rupture Consequently, this results in cell disintegration, paralysis of the intestines, cessation of feeding, and ultimately, the death of the insect.

Figure 2.2b Mechanism of action of toxins

(J Li, D J Derbyshire, B Promdonkoyt and D J Ellart (2001), Structural - endotoxin from implications for transformation of Bacillus thuringiensis.water-soluble to membrane –inserted forms, Biochemical Society: pp 571- 577.)

History of Bt and its commercial applications

Bt bio-pesticides offer a sustainable alternative to chemical pesticides, promoting environmental health and safety for humans Their effectiveness spans various applications, including agriculture, forestry, mosquito management, and general pest control.

“Bacillus bombyces” was identified by Louis Pasteur when he found that it could infect silkworms and cause sickness While studying mulberry disease in

In 1901, Japanese scientist Sigetane Ishiwata discovered the bacterium Bacillus thuringiensis (Bt), initially linked to the "sotto" disease affecting silkworms This bacterium was harmful as it caused silkworms to perish before maturing E Berliner later identified the same bacterium from the Mediterranean chalk moth larva, Anagasta kuehniella, in 1911 By 1915, Bt was scientifically named Bacillus thuringiensis after being found in a grain mill in Thuringia, Germany Since the 1950s, Bt products have been utilized, particularly strain HD-1 (Bt subsp Kurstaki), to effectively control Lepidoptera in the southern woodlands of the United States The toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A produced by Bt significantly reduce the reliance on chemical pesticides.

Microbiologists have identified bacterial strains with a recombinant gene that enables the expression of the Cry protein, enhancing pest management in agriculture through genetic modification Bt pesticides are known for their effectiveness in controlling insect pests; however, studies indicate the development of resistance to Bt toxins in Bacillus thuringiensis, leading to decreased efficacy against these pests Additionally, Bt preparations primarily target surface-dwelling worms on plants, limiting their overall impact.

Stem borers, which infest the stems of plants, can be effectively controlled However, environmental factors such as sunlight can quickly deactivate Bt preparations Consequently, transgenic plants have been developed and are now widely used worldwide The first transgenic plant, tobacco, was created in 1983 using Agrobacterium tumefaciens (Hernstadt et al., 1986) A few years later, the Cry gene, which produces a protective protein for crops like tomato, tobacco, and cotton, was identified in plants (Barton et al., 1987).

Vietnam, located in the northern subtropical hemisphere, boasts a diverse subtropical climate with various climatic zones, making it a potential reservoir for Bt genes Research indicates that Southeast Asia hosts a highly diverse population of Bt, characterized by significant serological diversity and soil dispersion This suggests that the natural environments of tropical and subtropical countries like Vietnam are abundant sources of Bt.

1971 saw the first application of the microbial insecticide Bt at the Plant

The Protection Institute in Vietnam, led by pioneers Nguyen Cong Binh, Pham Ba Nha, and Ngo Dinh Binh, has been at the forefront of Bt research In 1973, the Institute of Biology initiated manual and semi-industrial manufacturing of Bt in their laboratories, marking a significant advancement in the field.

From 1973 to 1976, Bt was primarily cultivated on a solid medium made from a handmade agar base, which included seaweed, peanut meal, soybean meal, and fish meal, yielding positive results for vegetables in the Hanoi suburbs In 1975, the Experimental Biology Department in Ho Chi Minh City developed Bt preparations through submerged fermentation in a homemade fermenter By 1982, the Food Industries Research Institute advanced this process, creating Bt inoculants using 5m³ capacity fermenters This period marked a significant advancement in the manufacture and effective use of Bt preparations, leading to fruitful research initiatives In recent years, the applications of Bt have expanded, with several organizations, including the Institute of Biotechnology, actively engaged in Bt research.

(IBT), Plant Protection Research Institute, Viet Nam Institute of Post Harvest Technology, and Food Industries Research Institute…

Overall, the following findings suggest that the variety of Bt strains in

Vietnam has a vast diversity of Bt strains, with significant increases over the years In 2005, Ngo Dinh Binh reported 1,080 identified Bt strains, which surged to 3,500 strains by 2010 Furthermore, T Pullaiah's book "Global Biodiversity: Volume 1: Selected Countries in Asia" indicates that the total number of Bt strains in Vietnam reached 7,364.

Researchers in Vietnam have made significant advancements in biotechnology by cloning and expressing genes from Bt strains Ngo Dinh Binh et al successfully cloned genes for Cry1C and Cry1D proteins from Bt aizaiwai strains found in soil samples from Hanoi and Ha Tinh, resulting in recombinant proteins that exhibited superior insecticidal activity compared to controls In 2000, Vo Thi Thu et al focused on the cry4A gene, which targets mosquito larvae for effective mosquito control Additionally, in 2003, Le Thi Thu Hien developed a transfer vector for the cry1A gene, enabling its introduction into cotton plants.

Since the late 20th century, Vietnam has focused on transferring insect resistance genes into plants to create pest-resistant varieties Research has included the transfer of the cry1A gene into crops like mung bean, broccoli, and cabbage using Agrobacterium tumefaciens (Khuat Huu Thanh, 2003; Dang Trong Luong et al., 1999) In 2003, Phan Dinh Phap and colleagues utilized a gene gun to introduce the cry1B gene into rice, and in 2005, the insect-resistant gene was successfully introduced into eggplant using Agrobacterium tumefaciens.

Bt Vietnam exhibits a diverse array of strains, protein crystal structures, and protein-coding genes, showcasing a wide range of activities Furthermore, Bt is widely distributed across the entire territory of Vietnam Increasingly, Vietnamese researchers have conducted and published studies, both domestically and internationally, on the isolation and application of Bt in various provinces.

 Commercial products, production and application

Conventional Bt products primarily target lepidopteran pests in agriculture and forestry, but recent developments have introduced Bt strains effective against coleopteran pests Additionally, in public health initiatives, Bt strains are utilized to combat dipteran vectors responsible for spreading parasitic and viral diseases.

Commercial Bt formulations serve as effective insecticides that can be applied to various surfaces, including foliage, soil, aquatic environments, and food storage facilities Once introduced into an environment, the vegetative cells and spores of Bt subspecies can persist as part of the natural microflora for extended periods, ranging from weeks to years, although their quantities diminish over time.

In 2010, the global area planted with Bt GM cotton reached 19.6 million hectares, marking an increase of 4.6 million hectares from the previous year The primary producers of Bt GM crops include the United States, Argentina, India, and Canada, which collectively account for 90 percent of the global Bt GM crop area By the end of 2013, the total area planted with genetically modified crops expanded to 28.8 million hectares From 1996 to 2012, the projected value of Bt transgenic crops was estimated at $68.9 billion, constituting sixty percent of the total value of GM crops worldwide In 2012 alone, the value of GM crops was approximately $12 billion, compared to a global total of $18.7 billion (James, C 2010).

Since the introduction of Bt crops, approximately 198 varieties across eight different crops, such as potato, soybean, corn, eggplant, cotton, rice, poplar, and tomato, have been developed Notably, maize has garnered the highest number of permits, totaling 137, from over 35 countries (ISAAA 2016).

Research for identification of Bacillus thuringiensis

Conventional methods for the identification of Bacillus thuringiensis include characterization of colonies and evaluation of morphological and biochemical characteristics

 Typical colonies of B thurigiensis on LB medium are round or flattened, milky white, colony size 1 mm - 7 mm

 Gram stain: B thuringiensis is a Gram-positive, rod-shaped, purple bacillus

 Coomassie brilliant blue stain: colonies are rod-shaped, slightly obtuse tips arranged in chains or individually; spore central ovoid; Poisonous blue crystals, oval, rhombic or triangular, varying in size

 Positive reaction for Esculin usability, Lecithinase production, Salicin fermentation, Sucrose fermentability

Traditional PCR screening of B thuringiensis collections has limitations in uncovering new cry genes, leading to the development of various PCR-based methods to detect and characterize known cry genes In 1993, Kalman et al introduced a technique for identifying variations within the cry1C group, utilizing a set of primers designed to anneal across the cry1Ca1 sequence, a method referred to as "PCR walking."

Two innovative PCR-based methods were developed to detect both known and potential novel cry genes These methods were adapted to cover a wider range of genres rather than focusing on a single set By performing multiple alignments of DNA coding sequences from various subfamilies, researchers identified conserved regions unique to each subfamily This characteristic facilitated the creation of 'universal' subfamily primers, leading to a new approach for investigating and analyzing the cry gene content in a B thuringiensis strain.

The initial method for discovering novel cry genes involved the combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) This two-step process utilizes group-specific primers and subsequent enzymatic digestion of the resulting amplicons Each gene is expected to produce a distinct RFLP pattern, reflecting the unique combinations of genes present.

Kuo and Chak (1996) and Juárez-Pérez (1998) employed various universal primers and restriction enzymes in their research However, the presence of more than four cry genes in a strain, commonly observed in wild-type strains, often results in a highly complex restriction profile, rendering analysis challenging.

Kuo and Chak (1996) faced challenges in distinguishing between the cry1Ca, cry1Cb, cry1Ea, and cry1Fa genes due to the strong similarity within the cry1 subfamily To address this, they proposed a method involving a second PCR cycle with alternate forward primers and an additional enzymatic reaction to amplify different gene portions This approach required extended electrophoresis to achieve higher resolution of restriction fragments When applied to standard strains and 20 wild B thuringiensis isolates, the method produced expected profiles for the standards and revealed unexpected variations in the wild isolates Cloning and sequencing of novel cry genes validated the effectiveness of this technique.

A second method for detecting novel cry genes is based on the use of two successive PCR reactions using specific and universal primers in a multiplex PCR (Juárez-Pérez V et al.,1997)

Chelliah et al (2019) conducted a study based on the transcriptional regulator gene and the crystal protein gene of B thuringiensis, as referenced by Porcar and Juárez-Pérez (2003) and Han et al (2006), to evaluate and compare the effectiveness of engineered biomarkers, including XRE, GroEl, and gyrB.

The GroEL gene, a housekeeping gene for the chaperonin protein, plays a crucial role in maintaining the relationship between bacteria and host cells This gene is specific to Bacillus thuringiensis, being present in all 50 strains studied (100%), and is also found in Bacillus cereus in 95% of cases (55 out of 58 strains) Additionally, it is detected in all strains of related B cereus groups, including B mycoides, B pseudomycoides, and B weihenstephanensis, with a 100% detection rate (3 out of 3 strains).

The gyrB gene encodes the e-subunit of DNA gyrase, an essential enzyme that regulates the helical structure of bacterial chromosomes, impacting processes such as replication, transcription, recombination, and repair This gene is uniquely present in Bacillus thuringiensis, with a 100% detection rate in examined strains, and is also found in Bacillus cereus (91% detection rate) and other members of the B cereus group, including Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis, which all show a 100% detection rate.

The XRE gene encodes a transcriptional regulatory protein characterized by a coding sequence (CDS) that features an HTH motif, which is common among the XRE-like protein family and MerR family transcriptional regulators These HTH proteins play a crucial role in various signaling pathways, including the inhibition of spore development and the regulation of proteolytic enzyme production Notably, the XRE gene has been shown to exhibit specificity in its functions.

Bacillus thuringiensis (43/50 of the 50 strains examined were positive, or 86%)

Meanwhile, just one strain of Bacillus cereus was identified (1/58, or 2%), and no strains of the other B.cereus group (B mycoides, B pseudomycoides, and B weihenstephanensis) were identified

PCR targeting Cry2 successfully amplified 6 strains of B cereus and 36 strains of B thuringiensis, accounting for 72% of the samples tested Notably, no amplification occurred for other B cereus group strains or non-B group strains, demonstrating an accuracy of 82% for Cry2 in detecting B cereus groups.

MATERIALS AND METHODS

Materials

All bacterial strains used in this study were presented in Table 3.1

Table 3.1 Bacterial strains used in the study

Standard Bt strain 4T1 Plant Protection

Veterinary Medicine - Vietnam National University of Agriculture

In this study, species-specific PCR was performed using four species- specific genes for Bt The primers for these genes were listed in Table 3.2

Table 3.2 PCR primers used in this study

Primer sequence 5' - 3' Product size (bp)

Location and time

 Research location: Laboratory of the Department of Plant Biotechnology - Faculty of Biotechnology - Vietnam National University of Agriculture.

Research contents

3.3.1 Identification of Bacillus thuringiensis by morphology and biochemical properties

Bacillus thuringiensis was isolated from microbial fertilizers using the method described by Travers et al (1987) The evaluation of the cell morphology, spores, and biochemical properties of the identified B thuringiensis colony was conducted following established protocols.

3.3.2 Determination of method for bacterial DNA extraction

The genomic DNA of Bt was extracted following three different methods including:

The CTAB method, as outlined by Farhad et al (2016), has been enhanced through modifications developed by the Laboratory of Plant Biotechnology at the Department of Plant Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture.

(3) Lysozyme-based method according to Mohsen et al (2016)

The quantity and purity of genomic DNA were measured by the Nano drop analyzer of Thermo Scientific and agarose gel electrophoresis

3.3.3 Identification of Bacillus thuringiensis based on species-specific PCR analysis

Determining the presence of specific genes using specific PCR primer

3.3.4 Confimation of Bacillus thuringiensis by crystalline protein staining

For the identification of B thuringiensis, crystal protein staining was performed as the method suggested by USFDA.

Research methodology

All strains were preserved in 40% glycerol stock at -30°C for long-term storage They were then streaked onto LB agar plates and incubated at 30°C overnight Following incubation, a pure colony was selected and inoculated into T3 agar medium at 37°C.

48 h These colony were used for subsequent experiments for identification utilizing different methodologies as depicted in Figure 3.1

Figure 3.1: Systemic representation of Bacillus thuringiensis identification

The study focused on the morphological characteristics of Bt isolates, including colony color, shape, elevation, and texture, alongside microscopic features such as Gram reaction, bacterial cell shape, and spore staining.

Confimation of Bacillus thuringiensis by crystalline protein staining

To prepare and fix slides for bacterial examination, use a sterile culture rod to collect a small amount of bacteria from the suspected colony on T3 medium Mix this with a drop of distilled water in the center of the slide and allow it to dry at room temperature To fix the slide, rapidly heat it over the flame of an alcohol lamp 2-3 times, ensuring the bacteria adhere firmly to the slide.

 Stain with crystal violet solution for 30 seconds to 1 minute, and rinse with water

 Stain Lugol solution for 1 minute, rinse with water

 Decolorize with 90% alcohol for 10-30 seconds: when seeing color loss, wash with water

 Stain with Fuschin solution for 1 min, wash with water, and air dry

- Examine the slide under the microscope's high-magnification oil objective

The differential properties of all the Bt isolates were examined for catalase, lecithinase and sucrose test according to Cinar et al (2008)

- After being grown on T3 agar medium, the single purified colonies of the

Bt isolates were placed on clean glass slides Apply a drop of 3% H 2 O 2 to the colony and observe the formation of air bubbles

- After being grown on T3 agar medium, the single purified colonies of the

Bt isolates were inoculated in 10% sucrose liquid medium supplemented with red phenol indicator and cultured at 30°C After 7 days, the color change of the bacterial culture medium was observed If:

- Positive reaction: the medium changes color from red-orange into magenta- pink

- Negative reaction: No color change of bacterial culture medium

- After being grown on T3 agar medium, the single purified colonies of the

Bt isolates were inoculated in MYP agar medium and incubated at 37°C for 18-24 hours The results showed the development of a white opaque and diffused zone that extended along the medium and surrounding colonies.

 Positive reaction: Precipitation around the streak of bacteria

The genomic DNA of Bt was extracted following three different methods including:

(1) CTAB method according to Farhad et al (2016) with modifications that were improved by Laboratory of Plant Biotechnology, Department of Plant Biotechnology, Faculty of Biotechnology, Vietnam National University Of Agriculture

(3) Lysozyme-based method according to Mohsen et al (2016)

 CTAB method extraction(Farhad et al.,2016)

After overnight incubation of the culture in liquid LB medium, achieving an OD600 range of 0.6 to 0.8, 2 ml of the culture was transferred to an Eppendorf tube and centrifuged at 10,000 g for 10 minutes at 4℃ The aqueous solution was then discarded.

- Preparation of extraction buffer: Dissolve CTAB (0.5g), EDTA (1g), Tris base (2.5g), and NaCl (5g) in 100ml of autoclaved distilled water, dissolve at room temperature

- The pellet was added 500 àL of CTAB solution xx àL βME and xx àL SDS 20% and crushed in 3 minutes using Mixer Mill MM 200 machine

- Step 1: Incubate the sample at 65℃ for 30 minutes and gently invert the tubes every 5 minutes

- Step 2: Add a mixture of Phenol:Chloroform:Isoamyalcohol (25:24:1) in a ratio of 1:1 (calculated according to the volume of buffer absorbed in step 2 compared to the mixture of 25:24:1)

- Step 3: Mix well to create a suspension, then centrifuge at 13700g at 4℃ for 10 minutes and collect the supernatant

- Step 4: Add the mixture of Chloroform: Isoamyalcohol (24:1) in a ratio of 1:1 (calculated according to the volume of buffer obtained in step 3 compared to the 24:1 mixture), mix well by vortex

- Step 5: Centrifuge at 13700g at 4℃ for 10 minutes, then take the supernatant into a new Eppendorf (1.5ml)

- Step 6: Add absolute ethanol with the ratio of 2 ethanol: 1 buffer obtained in step 6 (cold ethanol is better), and gently mix 6-8 times

- Step 7: Centrifuge at 10000g, at 4℃ for 7 minutes

- Step 8: Remove the supernatant and wash the DNA/RNA precipitated with

- Step 9: Centrifuge at 5400g, at 4℃ for 5 minutes, remove supernatant, and dry in air

- Step 10: Dissolve the precipitate in 50-100 àl of TE buffer and store it at - 20℃

To begin the process, take one colony from each isolated strain and immerse it in a PCR tube containing 100 µL of double distilled water Then, place the tube in the PCR machine and heat it to 95 °C for 5 minutes.

- Step 2 Take the PCR tube made in step 1, transfer the sample in the new 1.5ml effpendoff tube, centrifuge at 13000 rpm at 4 o C for 5 minutes

- Step 3 Supernatant containing genomic DNA was transfered in new tube and ready for subsequent PCR amplification

 Lysozyme and CTAB-based method

Follow Farhad Masoomi-Aladizgeh, Leila Jabbari, Reza Khayam Nekouei & Ali Aalami, Protocol Exchange (2016) and Mohsen Sohrabi et al.(2016) with minor modification

- After obtaining bacterial samples cultured in liquid LB medium with OD index from 0.6 to 0.8 To collect biomass, samples were centrifuged at 10000 g at 4℃ for 10 min

- Preparation of extraction buffer: Dissolve CTAB (0.5g), EDTA (1g), Tris base (2.5g), and NaCl (5g) in 100ml of autoclaved distilled water, dissolve at room temperature

To prepare the sample, 2 mL of an overnight culture grown in LB medium at 37°C for 20 hours was centrifuged at 10,000 rpm for 5 minutes After discarding the supernatant, the pellet was resuspended in 70 µL of TE (1:10) buffer Subsequently, 15 µL of Lysozyme (20 mg/mL) was added, and the mixture was incubated at 37°C for 30 minutes.

- Step 2: Crush the sample with 600 àL of CTAB solution, 1.2 àL βME, and 60àL SDS 20% in the cellbreaker

- Step 3: Transfer the resulting solution to a sterile centrifuge tube (size 2ml) Incubate the sample at 65˚C for 30 minutes, mix every 5 minutes during sample incubation for lysing cells completely

- Step 4: Add the mixture of Phenol: Chloroform: Isoamyl alcohol (25:24:1) in the ratio 1:1 (calculated by volume of buffer obtained in step 3 compared to the mixture of P: C: I)

- Step 5: Mix the sample by vortex and centrifuge at 13700 g, 4°C for 8 minutes Transfer the supernatant to a new tube (2 ml)

In Step 6, combine the Chloroform: Isoamyl alcohol mixture in a 24:1 ratio at a 1:1 volume with the buffer obtained in Step 4 Vortex the mixture thoroughly, then centrifuge it and transfer the supernatant into a new tube.

- Step 7: Centrifuge at 13700 g, 4˚C for 10 minutes Transfer supernatant to a new 1.5ml tube

- Step 8: Add ethanol 100% in the ratio 2 ethanol: 1 buffer (obtained in step

7) to precipitate the DNA, invert the tube 6-8 times to mix the solution

- Step 9: Centrifuge at 10000 g, 4˚C for 7 minutes

- Step 10: Pour the supernatant, wash the DNA/RNA precipitate with 500μl of 70% EtOH solution, shake vigorously several times

- Step 11: Centrifuge at 5400 g, 4˚C, 5 minutes, remove supernatant, drain the solution, and dry at room temperature

- Step 12: Dissolve the precipitate in 50 μl of TE buffer

Determination of total DNA concentration and qualification

To assess the concentration and purity of DNA in a sample, absorbance measurements between 230 and 320 nm are utilized The UV absorbance ratios A280/A260 and A230/A260 serve as indicators of DNA purity, with a quality DNA sample exhibiting an A260/A280 ratio between 1.7 and 2.0 Additionally, total DNA integrity is evaluated through agarose gel electrophoresis, where intact DNA appears as a distinct band, while degraded DNA manifests as multiple smaller bands or a smear of fragments.

DNA solution was diluted to 5 ng/àl for PCR reaction

To identify Bacillus thuringiensis (Bt) at the molecular level, species-specific PCR was conducted, amplifying four key genes: the housekeeping genes Chaperonin protein gene GroEL and topoisomerase gene gyrB, along with the transcriptional regulatory gene XRE1 and the crystal proteins gene Cry2.

Table 3.3 Components of PCR reaction

DNA template 5ng/àl 4 àl

 Mix the reaction mixture well with a pipette or vortex, followed by rapid centrifugation

 Note: The procedure should always carry out a test sample of the following samples in parallel:

+ Blank control: DNA template is replaced by water

+ The positive control: DNA of standard strain B thuringiensis 4T1

The PCR was run in ASTEC Thermal Cyclers (Gene Atlas, ASTEC, Japan) using a program as shown in Table 3.4 as below

 The annealing temperature (Ta) is: is indicated in Table 3.4 o Gene XRE: 54.9 0 C o Gene gyrB: 57.6 0 C o Gene GroEL: 62.2 0 C o Gene Cry2: 56.7 0 C

- Step 1 Weigh 2 grams of agarose into a heat-resistant glass jar

- Step 2 Pour 100 ml of 1X TAE solution, shake well

- Step 3 Put in the microwave, and heat until the agarose is completely dissolved

- Step 4 Prepare mold and comb, leveling

- Step 5 Allow the agarose to cool slowly to 60°C, add 5àl Ethidium Bromide to 100 mL of agar, shake well, and then pour gently and continuously into the mold, avoiding foaming

- Step 6 Allow the agarose to solidify (about 15 minutes), remove the comb, and place the gel mold in the electrophoresis bath

- Step 1 Fill the TAE 1X buffer with the electrophoresis tray

- Step 2 Cut a piece of paraffin, and aspirate 2μL of loading dye for each PCR product sample

- Step 3 Aspirate 7μL of PCR product solution, mix well with the sample buffer, and drop into the electrophoresis wells

- Step 4 Plugin the electrode so that the current flows from (-) → (+) from the well

- Step 5 Turn on the electrophoresis source fixed according to the voltage

(75 V) and amperage (200mA) for 55 minutes

- Preparation of slides and fixation: like Gram staining

- Stain with Coomassie brilliant blue solution for 3 minutes, wash with water, and dry

- Observe the shape of cells, spores, and crystals under the microscope at the oil objective

To identify B thuringiensis, crystal protein staining was conducted according to the method recommended by the USFDA The bacterial isolate was cultured on nutrient agar medium and incubated at 30 °C for 24 hours, followed by an additional incubation period of 5 days.

After incubating at room temperature, a bacterial smear was prepared, air-dried, and fixed through gentle heating The smear was then flooded with methanol for 30 seconds, followed by the application of 0.5% basic fuchsin, with gentle heating until steam appeared Finally, the slides were rinsed with water and examined under a microscope to identify the presence of tetragonal (diamond-shaped) toxin crystals.

Liquid LB medium buffered with 0.25 M sodium acetate:

Tryptone: 10 g Yeast extract: 5 g Sodium chloride: 5 g Sodium acetate: 20.5 g Distilled water: add just enough 1L

Distilled water: add just enough 1L

Distilled water: add just enough 1 L

Distilled water: add just enough 1 L

Sucrose fermentation medium: 10% sucrose solution, Red Phenol Coomassie brilliant blue solution

The culture mediums were autoclaved at 121 o C, 20 minutes

Medium and chemical of electrophoresis

- Electrophoresis running buffer, TAE 1x solution

RESULTS AND DISCUSSION

Identification of Bacillus thuringiensis by morphology and

Colonies from 6 isolated Bt (V, S, B, E, DT, T1) grown on T3 medium were observed for colony morphology and compared to the standard Bt strain (4T1)

Microscopy examination of T3 solid medium revealed a uniform colony morphology across all Bt isolates, characterized by flat, large, white colonies with diameters ranging from 1 to 4 mm, similar to the standard strain 4T1 Detailed descriptions of each Bt isolate's colony are provided in Table 4.1 Additionally, the colony morphology exhibited typical features of B thuringiensis on HiCrome Bacillus agar, confirming that all isolates belong to the B thuringiensis species (Alippi et al., 2019).

Table 4.1 Colony morphological characteristics of 6 Bt isolates on T3 solid medium

Number Isolated strains Description of the colony

1 4T1(Standard strain) Colonies are round, 1-2 mm in size, milky white, with thick centers and thin edges

2 S Colonies are round, 2-3 mm in size, milky white, centered, and rough surface

3 T1 Colonies are round, 1-2 mm in size, milky white, with thick wrinkled edges

4 DT Colonies are round, 3-4 mm in size, milky white, smooth surface, and thick center with a thin border

5 V Colonies are round, 2-3 mm in size, milky white, with wrinkled edges, with thick centers with thin edges

6 E Colonies are round, 2-3 mm in size, with a thick center with a thin border

7 B Colonies are round, 2-3 mm in size, with wrinkled edges

Figure 4.1 Morphology of B thuringiensis colonies isolates on T3 solid medium

- V, S, B, E, DT, T1: Bt isolates from biopesticides

Gram staining with Fushsin dye and subsequent microscopy were employed to determine the cell morphology of each Bt isolate As shown in Figure 4.2, the morphological characteristics of the six Bt isolates indicated that all were Gram-positive, rod-shaped bacteria.

Figure 4.2 Cell morphological characteristics of 6 Bt isolates

The staining images reveal that six Bt isolates exhibit cell morphology akin to the standard Bt strain (4T1), characterized by violet-colored, rod-shaped vegetative cells This observation indicates that all the Bt isolates are classified as gram-positive bacteria.

34 all strains have similar characteristics such as slightly obtuse heads arranged in chains or individually; sporangium ovoid, green; cytoplasm becomes red-pink

Many closely related bacterial species, including B cereus and B mycoides, display similar characteristics Additionally, Brevibacillus brevis and Brevibacillus laterosporus also show comparable culture traits.

HiCrome Bacillus agar (Alippi et al., 2019), thus making the proper identification ambiguous Therefore, further analysis is needed to proper and acurate identification of the bacterial isolates

Colonies of strains were cultured in LB medium for 24h and at 30°C After that, the colonies were tested for biochemical reactions

Lecithinase reactions produce distinct rings on the medium, varying in size and shape based on the strain Additionally, the culture medium of the strains exhibits a lighter color compared to the original.

The positive reaction (+) shows a ring of lecithinase precipitation around the colony of isolates

Catalase reaction results After adding H 2 O 2 to the strains, strong and fast bubbles appeared, the bubbles were milky white, the amount of bubbles varied between strains

The positive reaction (+) shows the generation of air bubbles

The results of sucrose fermentation reveal noticeable reactions, particularly in the color change of the medium as the pH shifts to acidic, transitioning from red-orange to magenta-pink Additionally, the density of the medium remains consistent before and after the reaction, closely resembling that of the original strain.

Positive reaction (+): the medium changes color from red orange into magenta-pink

Table 4.2 Biochemical test results of 6 isolates of Bt bacteria

(A) isolate V, (B) isolate DT, (C) isolate S, and (4T1) standard strain Bt colonies on T3 medium were cultured in dots on MYP agar and cultured at

To isolate V, DT, S, and standard strain (4T1), putative Bt colonies were transferred onto T3 medium Subsequently, two drops of 3% H₂O₂ were applied to the bacteria on the slide, resulting in the observation of air bubble formation.

Figure 4.3c Results of sucrose fermentation

(A) Negative control, (B) standard strain 4T1 (C) isolate V.Take the colonies growing above T3 into sterile 10% sucrose solution supplemented with phenol red and observe the results

All biochemical test results indicate that the Bt isolates closely resemble the standard Bt strain 4T1 These findings highlight the unique characteristics of Bt bacteria compared to other bacterial species exhibiting similar biochemical activities within the same genus.

Bacillus (Baumann et al., 1984; Claus & Berkley, 1986; Slepecky & Hemphill,

1992; Carlson & Kolstứ, 1993; Hansen et al., 1998) Therefore, it is possible to initially confirm that the 6 Bt isolate is belong to Bacillus genus

The colony and cell characteristics, along with biochemical reactions and spore-forming ability, confirm that all Bt isolates are Bacillus thuringiensis However, B cereus exhibits similar colony morphology and biochemical traits, necessitating molecular characterization using species-specific biomarkers for accurate identification (Alippi et al., 2019).

Determination of method for baterial DNA extraction

Molecular techniques in bacteriology begin with the extraction and purification of bacterial DNA Numerous DNA extraction methods have been developed, each offering distinct advantages and disadvantages Many of these techniques are derived from the traditional phenol-chloroform extraction method, which requires a varying number of reagents.

Numerous studies have focused on simplifying bacterial DNA extraction and purification methods, utilizing various lysing agents such as lysozyme, proteinase K, TWEEN20, and Triton X-100 (Gross-Bellard et al., 1973; Sambrook et al., 1989; Porteous et al., 1994; Agersborg et al., 1997; Zhu et al., 2005) Additionally, heat treatment of bacteria has proven to be an effective and straightforward approach for DNA extraction, facilitating successful applications in PCR and other molecular techniques (Dashti et al., 2009).

This study presents a straightforward and efficient approach for extracting total DNA from bacteria for PCR detection The genomic DNA of Bacillus thuringiensis (Bt) was extracted using three distinct methods: (1) the CTAB method as described by Farhad et al (2016), (2) a heat treatment method, and (3) a lysozyme-based method based on Mohsen et al (2016), with minor modifications applied to all Bt isolates and non-Bt groups.

Determination of purity of DNA

After extraction of the entire DNA, the product concentration and purity of the DNA were determined The A260/A280 ratio of the separation methods is shown in table 4.3

Table 4.3 OD 260 / 280 index results and concentration of 7 isolates

Lysozyme and CTAB-based method 254 1.96 2.31

Lysozyme and CTAB-based method 409 2.05 2.12

Lysozyme and CTAB-based method 105 1.90 2.22

Lysozyme and CTAB-based method 69 1.85 1.92

Lysozyme and CTAB-based method 136 2.00 2.33

Lysozyme and CTAB-based method 125 2.10 1.91

Lysozyme and CTAB-based method 108 1.99 1.86

The methods give OD ratios ranging from 0.98 to 2.10 with OD 260 /280 there is a big difference between the methods The method for the purest DNA is Lysozyme

The CTAB-based method, along with the 39 method, yields the most effective results for purified DNA, while the heat treatment method produces the least favorable outcomes, highlighting its limitations.

Determination of total DNA concentration

The extracted total DNA was analyzed for size using gel electrophoresis on a 2% agarose gel, with the results displayed via a gel scanner and documented through photographs (Figure 4.4a, 4.4b, 4.4c).

Figure 4.4a Heat treatment method for bacterial DNA extraction

V, E, S, B, T1, DT: Bt isolates ; DNA standard scale 100bp and 4T1( standard strain)

Figure 4.4b CTAB method for bacterial DNA extraction

V, E, S, B, T1, DT: Bt isolates ; DNA standard scale 100bp and 4T1( standard strain)

Figure 4.4c Lysozyme and CTAB-based method for bacterial DNA extraction

V, E, S, B, T1, DT: Bt isolates ; DNA standard scale 100bp and 4T1( standard strain)

All three DNA extraction methods yield positive results in total DNA, with the Lysozyme and CTAB-based method showing the best indicators However, this method is time-consuming and requires a significant amount of expensive chemicals In contrast, the PCR heat treatment method is more efficient, saving time and resources while still providing satisfactory total DNA and PCR reaction quality, making it a highly practical option.

The methods differ not only in method and results, but also in time, as shown in Table 4.4

Table 4.4 Estimated time for bacterial DNA extraction

PCR heat treatment method extraction ~15-20

DNA extraction with addition of

Lysozyme and CTAB-based method

In conclusion, the heat treatment method proved to be a simple, efficient, and convenient approach for extracting Bt DNA, successfully utilized for DNA extraction from all bacterial strains in preparation for subsequent PCR analysis.

4.3 Identification of Bacillus thuringiensis based on species-specific PCR analysis

To verify the molecular identity of the Bt isolates as Bacillus thuringiensis, we analyzed the presence of specific genes, including GroEL, GyrB, XRE, and Cry2, using PCR methods (Chelliah et al., 2019) The amplification results are illustrated in Figures 4.5a-d.

The specificity of primers targeting the XRE, gyrB, GroEL, and Cry2 genes for Bacillus thuringiensis was assessed through PCR analysis This included testing non-Bt strains such as ATCC79530 (Bacillus subtilis) and NT 1.8 (Bacillus amyloliquefaciens).

25923 (S aureus), ATCC 85922 (E coli) The results revealed the specific

42 amplification of the ~246 bp fragment (XRE gene); ~ 600 bp (GroEL); ~221bp

The gryB gene and approximately 700 bp of the Cry2 gene were exclusively found in standard B thuringiensis strains (4T1, 4T4, 4D4) and all Bt isolates, while they were absent in the non-Bt group These findings indicate a strong specificity of the primer for B thuringiensis.

Figure 4.5a PCR product with primers specific for the XRE gene

Blank control: DW (distilled water); 4T1, 4T4 , 4D4: positive control; non-

Bt groups: ATCC79530 (Bacillus subtilis), NT 1.8 (B amyloliquefaciens), ATCC

25923 (S aureus ), ATCC 85922 (E.coli), (standard strain): And the isolates are V,

E, S, B, T1, DT: Bt isolates 100bp DNA ladder

The study found that all 10 strains of B thuringiensis exhibited a complete positive amplification response, while the non-Bt group showed no amplification, resulting in a 0% response rate.

4 strains 0/4 (0%) Hence, XRE primers, which indicates the sensitivity and specificity of the transcriptional regulator gene targeting primer

Figure 4.5b PCR product with primers specific for the gyrB gene

Blank control: DW (distilled water); 4T1, 4T4, 4D4: positive control; non-Bt groups: ATCC79530 (Bacillus subtilis), NT 1.8 (B amyloliquefaciens), ATCC

25923 (S aureus ), ATCC 85922 (E.coli), (standard strain): And the isolates are V,

E, S, B, T1, DT: Bt isolates 100bp DNA ladder

The study found that all 9 strains of B thuringiensis exhibited a complete positive response, with a 100% amplification rate In contrast, the non-Bt group showed no amplification, resulting in a 0% response rate.

4 strains 0/4 (0%) Hence, gyrB primers, which indicates the sensitivity and specificity of the gene gyrase topoisomerase

Figure 4.5c PCR product with primers specific for the GroEL gene

Blank control: DW (distilled water); 4T1, 4T4, 4D4: positive control; non-Bt groups: ATCC79530 (Bacillus subtilis), NT 1.8 (B amyloliquefaciens), ATCC

25923 (S aureus ), ATCC 85922 (E.coli), (standard strain): And the isolates are V,

E, S, B, T1, DT: Bt isolates 100bp DNA ladder

The study found that all 9 strains of B thuringiensis exhibited a complete positive response, with a 100% amplification rate In contrast, the non-Bt group showed no amplification, resulting in a 0% response rate.

4 strains 0/4 (0%) Hence, GroEL primers, which indicates the sensitivity and specificity of the gene for chaperonin protein

 Detection of B thuringiensis uing Cry2 gene

Amplification products of approximately 700 bp were obtained using the primers cry2 (Figure 4.5d) As shown in Figure 4.5d, when using PCR targeting cry2, 3 standard strains (4T1, 4T4, 4D4) and 6 Bt isolates (100%) were amplified

No non-Bt group was amplified

Figure 4.5d PCR product with primers specific for the Cry2 gene

Blank control: DW (distilled water); 4T1, 4T4 , 4D4: positive control; non-Bt groups: ATCC79530 (Bacillus subtilis), NT 1.8 (B amyloliquefaciens), ATCC

25923 (S aureus ), ATCC 85922 (E.coli), (standard strain): And the isolates are V,

E, S, B, T1, DT: Bt isolates 100bp DNA ladder

This study's results align with the findings of Wei et al (2019) and Chellian et al (2019), demonstrating the high specificity of the primers used for identifying B thuringiensis Notably, Chellian et al (2019) employed six selective genes in their research.

This study focuses on distinguishing Bacillus thuringiensis from the Bacillus cereus group by analyzing specific genes, including lipoprotein-lipo, methyltransferase (MT), S-layer homology domain protein (BC), flagellar motor protein (motB), transcriptional regulator (XRE), and crystal protein (cry2), alongside two housekeeping genes, chaperonin protein (GroEL) and topoisomerase enzyme (gyrB).

Confirmatory identification of Bacillus thuringiensis by crystalline

Determination of the ability to produce toxic crystals and spores of putative Bt strains

Bacillus thuringiensis is known for its selective production of insecticidal crystal proteins, which serve as effective markers for identification In this study, PCR analysis revealed 700 bp bands corresponding to the cry2 gene in six Bt isolates and standard isolates, indicating the expected presence of crystal proteins across all tested Bt isolates.

To evaluate the production of toxic crystals and spores, colonies grown on T3 medium were assessed for endospore production using Coomassie brilliant blue solution and microscopic observation The findings regarding the spores and crystals of all Bt isolates are illustrated in Figure 4.7.

Description of crystals and spores

The isolates showed crystal and spore characteristics like those of standard

- Crystals are rhombic, round, eccentric, and many other shapes Full-color capture

- Cylindrical or ovoid spores, one end is colored, and the other is non- staining, whether internal or external

- Cells are entirely stained and cylindrical

Figure 4.7 Spores and crystals of some Bt isolates

The crystal protein staining results revealed the presence of crystal proteins in six Bt isolates, demonstrating similarities in crystal shape and spore characteristics when compared to standard Bt strains.

49 isolates produced rhombohedral crystals - the most common shape of crystals protein of Bt

Collectively, all the results (morphological and biochemical characteristics, species-specific PCR, and crystal protein staining) affirmed 6 isolates as B thuringiensis

CONCLUSIONS AND PROPOSALS

Conclusions

Morphological and biochemical analyses of six Bt isolates revealed a consistent colony type that is gram positive These isolates exhibited positive results in lecithinase, sucrose, and catalase tests, and they formed spores characteristic of Bacillus species, particularly Bacillus thuringiensis.

(2) Heat treatment method appeared to be simple, efficient and convinient method for bacterial DNA extraction

(3) Species-specific PCR analysis with XRE, GroEL, GyrB and Cry2 gene show high specificity towards B thuringiensis

(4) There was the presence of crystal proteins in 6 Bt isolates using crystal protein staining, affirming 6 isolates as B thuringiensis

Proposals

 Futher biochemical tests should be included

 Develop a multiplex PCR assay for identification of Bacillus thuringiensis

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APPENDIX Appendix 1 Catalase test results of six strains

Appendix 2 Test results on sucrose fermentation medium of six strains

Appendix 3 Lecithinase test results of six strains